Ostryopsis Intermedia, a New Species of Betulaceae from Yunnan, China

Botanical Studies (2010) 51: 257-262. SYSTEMATICS

Ostryopsis intermedia, a new species of Betulaceae from Yunnan, China

Bin TIAN, Teng-Liang LIU, and Jian-Quan LIU*

Molecular Ecology Group, MOE Key Laboratory of Arid and Grassland Ecology, School of Life Science, Lanzhou University, Lanzhou 730000, Gansu, P.R. China

(Received January 16, 2009; Accepted September 29, 2009)

ABSTRACT. Ostryopsis intermedia B. Tian & J. Q. Liu, a new species of Betulaceae from northwest Yun- nan, China, is described in this study. Morphologically, the new species shows a combination of traits (stable in all examined individuals and populations) of the two recognized species of the genus, which are reproduc- tively isolated from them by differences in flowering phenology and/or geographical distances. In addition, no intraspecific variation was found in the three species’ ITS sequences, but there were clear interspecific differences. These results collectively suggest that O. intermedia is sufficiently distinct from the other two described species of Ostryopsis to be recognized as a new species.

Keywords: Ostryopsis; Ostryopsis intermedia; New species.

INTRUDUCTION MATERIAL AND METHODS

Ostryopsis Decne. (Betulaceae) is endemic to China and In the morphological comparisons we examined at least is represented by two species: O. davidiana Decne., which 10 living individuals or herbarium specimens from each is widely distributed in northern China; and O. nobilis Bal- of the investigated populations to assess both interspecific four & Smith, restricted to Sichuan and northwestern Yun- morphological differences and the morphological stabil- nan (Li and Skvortsov, 1999). This genus has been placed ity of the putative new species and two known species at together with Carpinus L., Corylus L., and Ostrya Scopoli the population level. The results are shown in Table 1. In in the Coryloideae, largely based on the lack of a peri- addition, we recorded the flowering and fruiting phenol- anth in the staminate flowers (Thorne, 1992; Mabberley, ogy of the three species, by monitoring 10 individuals 1997). However, relationships among these genera remain from each of at least three populations of each species at unresolved, particularly concerning the phylogenetic posi- least once per month from March to August in 2007 and tion of Ostryopsis (Chen et al., 1999; Yoo and Wen, 2007). 2008. We also outlined the distributional range of each In the present study, we report a new species of this species according to field investigations and specimen endemic genus (Figure 1). During an extensive inves- records. Woodworth (1930) reported that chromosome of tigation of most populations of the genus over its en- O. davidiana is 2n = 16. In order to count the chromosome tire distribution range, we found a few populations in numbers of the three species, seeds were collected from northwest Yunnan previously ascribed to O. nobilis that their natural populations and germinated in petri dishes were morphologically different from the two known lined with moist gauze. The root tips were incubated in species of the genus. In order to evaluate whether these colchicine (0.1% w/v) for 2-3 h when they were 0.5 cm anomalous populations represent a new species, morpho- long, fixed in Carnoy’s fluid (absolute alcohol:glacial ace- logical traits, ITS sequences and the flowering phenology tic acid; 3:1) at 4°C for at least 30 min, hydrolyzed in 1 of representatives of these populations were analyzed mol/L hydrochloric acid at 60°C for 10 min, then washed and compared to those of the parapatrically distributed with water, stained with carbol fuchsin and squashed for O. nobilis and the geographically more isolated species, observations. The chromosome numbers were determined O. davidiana. The results collectively suggest that those from observations of 10 somatic cells. morphologically anomalous populations are sufficiently Three representative trees from each of three distantly distinct to be recognized as a new species. distributed populations of each species were randomly chosen for molecular (ITS sequence) identification and *Corresponding author: Email: [email protected], comparison (Table 2). Leaves of the selected trees were [email protected]; Fax: +86-931-8914288; Tel: collected and immediately dried in silica gel until total ge- +86-971-8914305. nomic DNA was isolated from them following the CTAB 258 Botanical Studies, Vol. 51, 2010 method of Doyle and Doyle (1987). The ITS regions of as an outgroup to root all sequenced individuals. The phy- nrDNA were amplified with ITS primers 1 and 4 (White logenetic relationships of the three species were assessed et al., 1990), then the PCR products were purified using by maximum likelihood (ML), Neighbor-Joining (NJ) and a TIANquick Midi Purification Kit following the sup- maximum parsimony (MP) analyses using PAUP* 4.0b10. plier’s recommended protocol (TIANGEN). Sequencing All gaps (indels) were coded as binary states (0 or 1). ML reactions were performed with the PCR primers to cover exhaustive search parameters were: simple addition of se- the whole PCR segment using an ABI Prism BigdyeTM quences of taxa with TBR branch swapping, MULTREES Terminator Cycle Sequencing Ready Reaction Kit. The and COLLAPSE options on. MP analyses (equally weight- reaction products were analyzed using an Applied Biosys- ed characters and nucleotide transformations) involved a tems model 3130xl automated sequencer (Perkin Elmer heuristic search strategy with 100 replicates of random ad- Applied Biosystems). All sequences were submitted to dition of sequences, in combination with ACCTRAN char- GenBank (accession numbers: GQ250099 to GQ250101), acter optimization, MULPARS+TBR branch swapping and then aligned using ClustalX version 1.81 (Thompson et al., STEEPEST DESCENT options on. In these analyses we 1997). Carpinus turczaninowii (AF081518) was selected used bootstrap values (BS) to assess branch support.

Figure 1. Ostryopsis intermedia B. Tian & J. Q. Liu A, fruiting branch; B, fruit; C, seed. Drawn by A. L. Li from B. Tian & J. Q. Liu 2007003 (LZU). TIAN et al. — Ostryopsis intermedia, a new species 259

RESULTS No intraspecific variation in ITS sequences was found among the nine examined individuals of each species The new species (Ostryopsis intermedia) differs from (three from each of three different populations). However, the two known species of the genus, O. nobilis and O. all these sequences are species-specific, with distinct in- davidiana, in a number of morphological characters terspecific differences, including a total of eight mutations (listed in Table 1). All these characters are stable within and three indels (Table 3). The new species differs from each species. The habit and leaf shape of O. intermedia O. nobilis at nine ITS sites, and from O. davidiana at just are similar to those of O. nobilis, except that the new three sites (pairwise differences: 1.5% and 0.5%, respec- species has doubly serrate leaves with acuminate or acute tively). All phylogenetic analyses produced a similar to- apices (Figure 1), while O. nobilis has irregularly serrate pology with high bootstrap supports for all nodes (100% leaves with obtuse or rounded apices (Table 1). Ostry- in all analyses), indicating that the new species is sister to opsis intermedia flowers and sets fruits early, from April the northerly species, O. davidiana (Figure 4). to May, while both O. nobilis and O. davidiana start to flower in June or July (Table 1). Geographically, the new DISCUSSION species is found in southwest China and parapatric to O. nobilis, but distantly isolated from O. davidiana (Figure The results of our molecular investigation suggest that 3). In the only known sympatric site, the new species and the new species is a sister species to O. davidiana, and O. O. nobilis showed no overlapping of flowering and fruit- nobilis is sister to the clade composed of O. davidiana and ing periods (Table 1). More than 1000 mature individuals the new species. Ostryopsis davidiana and O. intermedia were found at each of the six recorded sites, and more than share several morphological characters, including acumi- half of them can normally flower and set seeds. The total nate or acute leaf apices, doubly serrate leaf margins, and distribution range of the new species is similar to that of capitate infructescences. However, O. intermedia differs O. nobilis, and both have limited distributions (Figure 3), from the northern species in having larger, more densely although the range of O. davidiana (in northern China) is pubescent leaves that lack resinous glands. The new spe- more extensive. All three species were found to have the cies resembles O. nobilis in leaf size, habit and habitat; the same chromosomes number of 2n = 16 (Figure 2). main differences between them are in the characters shared

Table 1. Morphological and phenological differences between Ostryopsis intermedia and two known species. Character O. davidiana O. intermedia O. nobilis Leaf blade Ovate or elliptic-ovate, rarely Broadly ovate or ovate-orbicu- Broadly ovate or ovate-orbic- broadly ovate or broadly ob- lar, margin doubly serrate and ular, rarely ovate, margin ovate, margin doubly serrate usually incised above middle, irregularly serrate, apex obtuse and usually incised above mid- apex acuminate or acute or rounded dle, apex acuminate or acute Adaxial leaves Sparsely white pubescent Densely white pubescent Densely yellow tomentose Leaf size 1.5-5×1-4 cm 3-9 × 2-6 cm 3-10 × 2-6 cm Leaf resinous gland Yes No No Infructescence Capitate Capitate Racemose Fruiting period July-August April-May June-July

Table 2. Locations of populations used for ITS sequence comparison. Taxon Location Lat. (ºN) Long. (ºE) Elevation (m) Ostryopsis davidiana Tangchang, GanSu 33º59’ 104º28’ 1680 Maoxian, SiChuan 31º39’ 103º48’ 1570 Lingyuan, LiaoNing 40º55’ 119º16’ 720 O. nobilis Lijiang YunNan 27º16’ 100º13’ 1910 Xianggelila, YunNan 27º48’ 99º28’ 1950 Muli, SiChuan 27º49’ 101º12’ 2190 O. intermedia Weixi, YunNan 27º13’ 096º04’ 1980 Lijiang, YunNan 27º16’ 100º13’ 1910 Deqin, YunNan 28º22’ 98º54’ 2870 260 Botanical Studies, Vol. 51, 2010

Table 3. Variable sites of the aligned ITS sequences between the three Ostropsis species. Variable positions Taxon 58 72 82 108 170-172 225 395 484 565 582 586 O. davidiana G C A C GT- C C G C A C O. intermedia - T A C GT- T C G C A T O. nobilis - - G T ACA C T C T G T

Figure 2. Mitotic chromosomes of O. intermedia and O. nobilis. A, O. intermedia (2n = 16); B, O. nobilis (2n = 16).

Figure 3. The distributional ranges (lines) and locations of the investigated populations (dots) of the three Ostyopsis species: O. davidiana (black), O. nobilis (white) and O. intermedia (gray). TIAN et al. — Ostryopsis intermedia, a new species 261 by the new species and O. davidiana (Table 1). Overall, the new species shows a combination of morphological traits (stable in all examined individuals and populations) of the two known species, O. davidiana and O. nobilis, and seems to be an intermediate taxon between them. Hybrids between two differentiated species also generally have intermediate traits between their parental species (Grant, 1981). However, rather than representing a stable and isolated evolutionary lineage (species), hybrid swarms usually show inter-individual morphological variations. In addition, hybrids are usually distributed intermediately between the two assumed parental species and generally have completely or partly overlapping flowering stages with both parents. In contrast, the distributional ranges of the three species studied here suggest that O. davidiana is geographically isolated from both the new species and O. nobilis (Figure 3); the new species is distributed in more Figure 4. The single most parsimonious phylogenetic tree southern regions than O. nobilis and although the new spe- constructed based on ITS data. Numbers above branches indicate cies is parapatric to O. nobilis (one known population oc- bootstrap supports. curs sympatrically with O. nobilis) neither their flowering nor fruiting periods overlap (Table 1, Figure 3). Common mechanisms of reproductive isolation include terminal, racemose-capitulate; peduncle 2-3 mm, densely auto- and allo-polyploidy, which usually lead to immedi- white tomentose; infructescence capitate, bracts forming ate isolation from ancestral lineages and strongly promote a tubular sheath, ca. 1-1.5 cm, leathery, densely white pu- maintenance of the new evolutionary make-up when the bescent, striate, 3-lobed at apex, dehiscent on 1 side when new and ancestral lineages have sympatric or parapatric mature. Nutlet brown, ovoid or subglobose, 4-6 × 4-5 mm, distributions (Grant, 1981). However, this new species shiny, sparsely pubescent, ribbed; Fl. Mar - Apr, fr. Apr- cannot have arisen by polyploidy since all three of the May; Chromosome number: 2n = 16. species are diploid (Figure 2). Understanding more about Thickets on sunny mountain slopes; 1,500-2,500 m. the origins and histories of these species is a target of our NW Yunnan. future research. In addition, since the new species and O. nobilis have limited distributions and wild populations are Key to three species of Ostryopsis sparse, both of them warrant high priority for protection 1. Leaf adaxially densely yellow-brown tomentose, margin for further scientific research. irregularly serrate, apex obtuse or rounded; infructescence racemose...... O. nobilis TAXONOMIC TREATMENT 1. Leaf adaxially white pubescent, margin doubly serrate and usually incised above middle, apex acuminate or Ostryopsis intermedia B. Tian & J. Q. Liu, sp. nov.— acute, infructescence capitate. Type: CHINA. Yunnan: Lijiang, Judian, in thickets on 2. Leaf 1.5-5×1-4 cm, ovate or elliptic-ovate, rarely sunny mountain slopes, alt. 1,910 m, 2007-05-09, B. broadly ovate or broadly obovate, adaxially sparsely Tian & J. Q. Liu 2007003 (holotype, LZU; isotype, white pubescent, with yellow or brown resinous LZU). Figure 1 glands...... O. davidiana Species O. nobilis Balfour & Smith. affinis, sed foliis 2. Leaf 3-9 × 2-6 cm, broadly ovate or ovate-orbicular, apice acutis, margine duplicato-serratis, infra dense pu- rarely ovate, adaxially densely white pubescent, bescentibus albus; infructescentiis capitula differtis. Etiam without yellow or brown resinous glands...... similis O. davidiana Decne, quae foliis infra sparse pubes- ...... O. intermedia centibus et glandulosis. Shrubs to 5 m tall. Branches brown or gray, glabrous; Acknowledgements. This study was supported by grants branchlets brown, densely gray pubescent, sometimes from the National Natural Science Foundation of China with red-brown seta. Petiole 2-5 mm, densely pubescent; (30725004) and Ministry of Education of China. We thank leaf blade broadly ovate or ovate-orbicular, 3-9 × 2-6 cm, Dr. Deng Yunfei to write Latin Summary and Miss Li Ai subleathery, abaxially sparsely white pubescent, adaxi- Li to draw figure 1. ally densely white pubescent, base cordate or obliquely cordate, margin doubly serrate and usually incised above middle, apex acuminate or acute; lateral veins 6-9 on each LITERATURE CITED side of midvein, raised abaxially. Male inflorescences 1 or 2; bracts densely white pubescent. Female inflorescence Chen, Z.D., S.R. Manchester, and H.Y. Sun. 1999. Phylogeny 262 Botanical Studies, Vol. 51, 2010

and evolution of the Betulaceae as inferred from DNA Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and sequences, morphology, and paleobotany. Am. J. Bot. 86: D.G. Higgins. 1997. The clustal_x windows interface: Flex- 1168-1181. ible strategies for multiple sequence alignment aided by Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation proce- quality analysis tools. Nucleic. Acids. Res. 25: 4876-4882. dure for small quantities of fresh leaf tissue. Phytochem. Thorne, R.F. 1992. Classification and geography of the flowering Bull. 19: 11-15. plants. Biol. Rev. 58: 226-257. Grant, V. 1981. Plant Speciation. Columbia Univ Press, New Yoo, K.O. and J. Wen. 2007. Phylogeny of Carpinus and sub- York. family Coryloideae (Betulaceae) based on chloroplast and Li, P.C. and A.K. Skvortsov. 1999. Betulaceae. In C.Y. Wu and nuclear ribosomal sequence data. Plant Syst. Evol. 267: P.H. Raven (eds.), Flora of China, Vol. 2: Science Press, 25-35. Beijing and Missouri Botanical Garden Press, St. Louis, pp. White, T.J., T. Bruns, S. Lee, and J.W. Taylor. 1990. Ampli- 284-313. fication and direct sequencing of fungal ribosomal RNA Mabberley, D.J. 1997. The Plant Book. 2nd ed. Cambridge Uni- genes for phylogenetics. In M.A. Innis, D.H. Gelfand, J.J. versity Press. Cambridge. Sninsky, and T.J. White (eds.), PCR Protocols: a Guide to Methods and Applications. Academic Press. San Diego, pp. Swofford, D.L. 2003. PAUP*: phylogenetic analysis using par- 315-322. simony (*and other methods), Version 4.0b 10. Sunderland, Massachusetts: Sinauer Associates. Woodworth, R.H. 1930. Cytological studies in the Betulaceae. 4. Betula, Carpinus, Ostrya, Ostryopsis. Bot. Gaz. 90: 108-115.

中國雲南樺木科一新種：居中虎榛子

田斌劉騰靚劉建全

蘭州大學生命科學學院乾旱與草地生態教育部重點實驗室分子生態研究所

本文報導樺木科虎榛子屬一新種：居中虎榛子。該新種形態上位於虎榛子屬已有兩個物種之間，但這一新的形態特徵組合在所有檢查的個體和居群中都十分穩定。該新種在開花物候以及地理上與已有的兩個物種存在生殖隔離。ITS 序列在種內不存在變異，種間區別明顯並具種特異性；新種與兩個已知種之間都存在顯著的 ITS 序列差異。所有的證據都表明應將居中虎榛子獨立為一個新種。

關鍵詞：虎榛子屬；居中虎榛子；新種。